Essential Role Of Bax, Bak, And Caspase-9 In Betulin-induced Human Cancer Cell Apoptosis

Apoptosis is an evolutionarily conserved form of cell suicide and requires a specialized proteolytic system involving a family of proteases called Caspases. Caspases are synthesized in cells as inactive zymozens and, upon stimulation by apoptotic signals,are processed into mature tetrameric forms with two large and two small subunits. Activated Caspases participate in the cleavage of a set of proteins, resulting in the disassembly of the cell. Two main Caspase activation cascades have already been described, One is initiated by the activation of cell-surface death receptors, such as Fas and TNF. leading to Caspase-8 activation, which in turn cleaves and activates downstream effector Caspases such as Caspase-3,-6,and-7. An alternative mitochondrial pathway is triggered by cytochrome c released from the mitochondria, which binds to the Caspase-activating protein Apaf-1,stimulating the binding of Apaf-1 to pro-Caspase 9 and inducing the processing and activation of this Caspase. The permeabilization of the mitochondrial outer membrane and the release of cytochrome c are regulated by Bcl-2 family proteins. The multi-domain pro-apoptotic molecules Bax and Bak serve as an obligatory gateway for cytochrome c release in response to diverse stimuli.Defects in apoptosis may contribute to tumor progression and resistance to treatment. Elucidatingthe molecular mechanisms associated with specific apoptotic processes, and thereby identifying means to trigger effective apoptosis, is an attractive therapeutic strategy to cure human cancers.Betulin (lup-20(29)-ene-3β,28-diol)is a naturally occurring triterpene and is abundantly found in mainly bushes and trees, forming the principal extractive (up to 30% of dry weight) of the bark of birch trees.It can be used as a starting compound for other useful chemicals.which possess various interesting pharmacological properties. Certain structure-related derivatives of betulin. particularly betulinic acid, have been demonstrated to be cytotoxic drugs and exhibit anti-tumor activity against many types of tumors.Recent studies reported the cytotoxicity of betulin, but the underlying mechanism was not elucidated. The aims of the present investigation were to determine whether betulin has type specific anti-cancer activity in human cancers and to explore the molecular mechanisms by which it triggers cancer cell death.In this study, we obtained the following results:1. Cytotoxic effect of betulin on various cancer cells. The results showed that betulin significantly inhibited cell viability in cervix carcinoma HeLa cells, hepatoma HepG2 cells, lung adenocarcinoma A549 cells.and breast cancer MCF-7 cells with IC50 values ranging from 10～15μg/mL.While betulin exhibited only moderate anticancer activity in other human cancer cells such as, hepatoma SK-HEP-1 cells. prostate carcinoma PC-3.and lung carcinoma NCI-H460. with IC50 values ranging from 20～60μg/mL. it showed minor growth inhibition in human erythrolukemia K562 cells (IC50>100μg/mL).2.Betulininduces apoptosis cell death in HeLa cells. We further investigated the mechanism of anticancer activity by betulin, using HeLa cells as an experimental model.Betulin (10μg/mL) induces apoptotic cell death, as evidenced by morphological characteristics such as membrane phosphatidylserine translocation, nuclear condensation/fragmentation, and apoptotic body formation. Furthermore, an immunoblotting analysis showed that Caspase-3 substrate PARP cleavage to yield the 85kDa version of theprotein occurred in HeLa cells 8 hpostbetulintreatment.3. Betulin-induced activation of Caspase-9,-3.The results showed that Caspase-9 activity was detectedin cells treated with betulin for 4 h and gradually decreased after that. The activation of Caspase-3 was detected post 8 h after treatment with botulin(10μg/mL).The Caspase-3 activity was elevated a further 4-fold after 12 h and 24 h of treatment with betulin.4.Apoptosis of betulin-induced HeLa cells is mediated through cytochrome c and Smac release.A kinetics analysis showed that the depolarization of mitochondrial membrane potential and the release of mitochondrial cytochrome c occurred as early as 30 min after treatment with betulin. Immunoblotting analysis demonstrated that cytochrome c and Smac in the cytoplasmic fraction appeared as early as 30 min post betulin treatment and gradually increased over time.5.Betulin-induced apoptosis in HeLa cells is associated with mitochondrial translocation of Bax and Bak. The mitochondrial proteins Bax and Bak serve as a necessary gateway for cytochrome c release at the mitochondrial outer membrane.Importantly, Bax and Bak were rapidly translocated to the mitochondria 30 min after betulin treatment.6.Reduced expression of Caspase-9 in SK-HEP-1 cell may contribute to its resistance to betulin-induced apoptosis.We examined the expression pattern of apoptosis regulatory proteins in two cell lines. HepG2 and SK-HEP-1,and found that the expression level of Caspase-9 in SK-HEP-1 cells is much lower than that in HepG2 cells.When we restore the expression level of Caspase-9 in SK-HEP-1 cells similar to that in HepG2 cells may enhance the sensitivity of cells to apoptosis.In conclusion, exploiting the mechanistic understanding of the selective cytotoxic effect of betulin on some cancer cells may aid in the development of novel and potent chemotherapeutic agents accompanied by diminished side effects.